Sulfotransferases comprise a family of enzymes that catalyze the transfer of a sulfonate or sulfuryl group (SO.sub.3.sup.31 ) from the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (1) to an acceptor molecule. Even though it is more accurate to call these sulfonation reactions, the term sulfation is still widely used. The term sulfation will be employed within this specification with the understanding that a sulfonate or sulfuryl group is transferred. Sulfotransferases mediate sulfation of different classes of substrates such as carbohydrates, oligosaccharides, peptides, proteins, flavonoids, and steroids for a variety of biological functions including signaling and modulation of receptor binding (Bowman, K. G., et al., (1999) Chem. Biol. 6, R9-R22; and Falany, C. N., (1997) FASEB J. 11, 1-2). Within the past three years, many new sulfotransferases have been identified and cloned (Aikawa, J.-I., et al., (1999) J. Biol. Chem. 274, 2690; Dooley, T. P., (1998) Chemico-Biol. Interact. 109, 29; Fukuta, M., et al. (1998) Biochim. Biophys. Act. 1399, 57; Habuchi, H., et al., (1998) J. Biol. Chem. 273, 9208; Mazany, K. D., et al., (1998) Biochim. Biophys. Act. 1407, 92; Nastuk, M. A., et al. (1998) J. Neuroscience 18, 7167; Ong, E., et al., (1998) J. Biol. Chem. 273, 5190; Ouyang, Y.-B., et al., (1998) J. Biol. Chem. 273, 24770; Saeki, Y., et al. (1998) J. Biochem. 124, 55; Uchimura, K., et al. (1998) J. Biol. Chem. 273, 22577; and Yoshinari, K., et al., (1998) J. Biochem. 123, 740). A facile means to produce large amounts of sulfated product and efficient sulfotransferase assays are essential for the biological study of these enzymes and their sulfated products.
Sulfotransferases are the family of enzymes catalyzing sulfotransfer reactions, or the transfer of a sulfuryl group (SO.sub.3) from 3'-phosphoadenosine-5'-phosophosulfate (PAPS) to an acceptor molecule. Sulfotransferases, present in most organisms and in all human tissues, mediate sulfation of different classes of acceptors for a variety of biological functions. To date, more than 30 sulfotransferase cDNAs have been isolated from animal, plant, and bacterial sources (Weinshilboum, R. M., et al. (1997) FASEB J. 11, 3-14). The varied and important roles sulfotransferases play in biological systems have only recently been uncovered, including detoxification, cell signaling, and modulation of receptor binding (Bowman, K. G., et al. (1999) Chern. Biol. 6, R9-R22; and Falany, C. N., (1997) FASEB J. 11, 206-216). Drug design to selectively inhibit these therapeutically important enzymes has quickly followed the discovery of their biological roles (Seah, V. M. Y., et al. (1994) Biochem. Pharmacol. 47, 1743-9; Bartzatt, R., et al. (1994) Biochem. Pharmacol. 47, 2087-95; Matsui, M., et al. (1995) Biochem. Pharmacol. 49, 739-41; Wong, C.-K., et al. (1997) Biochem. Biophys. Res. Commun. 233, 579-583; and Schuur, A. G., et al. (1998) Chem. Res. Toxicol. 11, 1075-1081). Given the monumental speed with which new sulfotransferases are being identified and interest in their synthetic application and the development of sulfotransferase inhibitors, simple and rapid activity assays are vital to the progress of this field.
PAPS, the universal sulfate donor and source of sulfate for all sulfotransferases, is a highly expensive and unstable molecule that has been an obstacle to the large-scale production of enzymatically sulfated products. The half-life of PAPS in aqueous solution at pH 8.0 is approximately 20 hours and is available from Sigma Co. Product inhibition by adenosine 3',5'-diphosphate (PAP) (3) has also been a limiting factor to large-scale applications. PAP inhibition of hydroxysteroid sulfotransferase was determined to be K.sub.i =14 .mu.M ( Marcus, D. J., et al. (1980) Aial. Biochem. 107, 296). PAP inhibits NodST with K.sub.i =0.1 .mu.M (Lin, C.-H., et al., (1995) J. Am. Cheni. Soc. 117, 8031).